Pretreatment Of Peritoneal Macrophages Research Articles

Activation of Cannabinoid Receptor 2 Ameliorates DSS-Induced Colitis through Inhibiting NLRP3 Inflammasome in Macrophages.

Activation of cannabinoid receptor 2 (CB2R) ameliorates inflammation, but the underlying mechanism remains unclear. In the present study, we examined whether activation of CB2R could suppress the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome. In peritoneal macrophages isolated from C57BL/6 mice, LPS/DSS challenge for 24 h increased the expression of the components of NLRP3 inflammasome NLRP3, Casp-1 p20/Casp-1 p45 ratio, proIL-1β and IL-1β and also enhanced autophagy (LC3-II/LC3-I ratio, Beclin-1 and SQSTM1). Pretreatment of peritoneal macrophages with HU 308, a selective CB2R agonist, attenuated LPS/DSS-induced NLRP3 inflammasome activation, but further enhanced autophagy. In comparison with wild-type (WT) control, peritoneal macrophages from CB2R knockout (KO) mice had more robust NLRP3 inflammasome activation and attenuated autophagy upon LPS/DSS challenge. Knockdown autophagy-related gene 5 (Atg5) with a siRNA in peritoneal macrophages attenuated the inhibitory effects of HU 308 on LPS/DSS-induced NLRP3 inflammasome activation in vitro. In vivo, HU308 treatment attenuated DSS-induced colitis mice associated with reduced colon inflammation and inhibited NLRP3 inflammasome activation in wild-type mice. In CB2R KO mice, DSS-induced inflammation and NLRP3 inflammasome activation were more pronounced than those in WT control. Finally, we demonstrated that AMPK-mTOR-P70S6K signaling pathway was involved in this CB2R-mediated process. We conclude that activation of CB2R ameliorates DSS-induced colitis through enhancing autophagy that may inhibit NLRP3 inflammasome activation in macrophages.

Sargassum fusiforme polysaccharide activates nuclear factor kappa-B (NF-κB) and induces cytokine production via Toll-like receptors

This study was designed to investigate the mechanism of macrophage activation by the Sargassum fusiforme polysaccharide (SFPS). As a result, SFPS significantly enhanced cytokines and nitric oxide (NO) productions in peritoneal macrophages, and stimulated macrophages to produce the cytokines and NO through the induction of their genes expression. The pretreatment of peritoneal macrophages with special antibodies [Toll-like receptors (TLRs) antibody] significantly blocked SFPS-induced tumor necrosis factor alpha (TNF-α) and NO production. Furthermore, pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB, effectively suppressed SFPS-induced TNF-α and interleukin 1β (IL-1β) secretion in peritoneal macrophages, indicating that SFPS stimulated macrophages to produce cytokines through the NF-κB pathway and the result was further confirmed by the experiment of Western blotting (WB) and confocal laser scanning microscope (CLSM). Taken together, these results suggest that SFPS-mediated induction of cytokines and NO production in macrophages is mediated, at least in part, by TLRs/NF-κB signaling pathway.

Effects of aging on endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

BackgroundPeriodontitis is a bacterially induced chronic inflammatory disease. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, termed endotoxin tolerance. Aging has a profound effect on immune response to bacteria challenge. The aim of this study was to explore the effects of aging on endotoxin tolerance induced by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and Escherichia coli (E. coli) LPS in murine peritoneal macrophages.Methodology/Principal FindingsWe studied the cytokine production (TNF-αand IL-10) and Toll-like receptor 2, 4 (TLR2, 4) gene and protein expressions in peritoneal macrophages from young (2-month-old) and middle-aged (12-month-old) ICR mice following single or repeated P. gingivalis LPS or E. coli LPS stimulation. Pretreatment of peritoneal macrophages with P. gingivalis LPS or E. coli LPS resulted in a reduction in TNF-α production and an increase in IL-10 production upon secondary stimulation (p<0.05), and the markedly lower levels of TNF-α and higher levels of IL-10 were observed in macrophages from young mice compared with those from middle-aged mice (p<0.05). In addition, LPS restimulations also led to the significantly lower expression levels of TLR2, 4 mRNA and protein in macrophages from young mice (p<0.05).Conclusions/SignificanceRepeated LPS stimulations triggered endotoxin tolerance in peritoneal macrophages and the ability to develop tolerance in young mice was more excellent. The impaired ability to develop endotoxin tolerance resulted from aging might be related to TLR2, 4 and might lead to the incontrollable periodontal inflammation in older adults.

Proteasome InhibitionIn VivoPromotes Survival in a Lethal Murine Model of Severe Acute Respiratory Syndrome

Ubiquitination is a critical regulator of the host immune response to viral infection, and many viruses, including coronaviruses, encode proteins that target the ubiquitination system. To explore the link between coronavirus infection and the ubiquitin system, we asked whether protein degradation by the 26S proteasome plays a role in severe coronavirus infections using a murine model of SARS-like pneumonitis induced by murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal macrophages with inhibitors of the proteasome (pyrrolidine dithiocarbamate [PDTC], MG132, and PS-341) markedly inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity in macrophages, as well as the induction of inflammatory mediators such as IP-10, gamma interferon (IFN-γ), and monocyte chemoattractant protein 1 (MCP-1). In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in A/J mice. Treatment of A/J mice with the proteasome inhibitor PDTC, MG132, or PS-341 led to 40% survival (P < 0.01), with a concomitant improvement of lung histology, reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and reduced pulmonary inflammatory cytokine expression. These data demonstrate that inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene expression in vivo by reducing MHV-1 replication and the resulting inflammatory response. The results further suggest that targeting the proteasome may be an effective new treatment for severe coronavirus infections.

Extracellular heat shock cognate protein 70 induces cardiac functional tolerance to endotoxin: Differential effect on TNF-α and ICAM-1 levels in heart tissue

Endotoxin provokes cardiac dysfunction, and induction of tolerance to endotoxin has therapeutic potential. Heat shock protein 70 (HSP70) can induce endotoxin tolerance in macrophages. We recently found that heat shock cognate protein 70 (HSC70) induces pro-inflammatory cytokines via activation of TLR4 in macrophages and the myocardium. We hypothesize that HSC70 preconditioning induces cardiac tolerance to endotoxin. Pretreatment of peritoneal macrophages with HSC70 for 24 h reduced TNF-α levels following endotoxin stimulation. Preconditioning of mice with HSC70 24 h prior to endotoxin attenuated endotoxemic cardiac dysfunction. HSC70 preconditioning reduced TNF-α levels in plasma and heart tissue by 33.3% and 35.4%, respectively, and decreased ICAM-1 levels in heart tissue by 63.5% following endotoxin challenge. The effect of HSC70 on TNF-α was less robust than endotoxin preconditioning (79.7% and 75.0% reduction in TNF-α levels in plasma and heart tissue, respectively); however, HSC70 and endotoxin preconditioning had comparable effects on ICAM-1 levels in heart tissue. While HSC70 preconditioning had no effect on myocardial TLR4 protein levels, it suppressed NF-κB activation induced by endotoxin. We conclude that HSC70 preconditioning (1) attenuates the TNF-α response to endotoxin in macrophages in vitro, (2) induces cardiac functional tolerance to endotoxin and (3) reduces NF-κB activity, and TNF-α and ICAM-1 levels in heart tissue. Thus, the mechanism of HSC70-induced cardiac tolerance to endotoxin appears to involve down-regulation of myocardial TLR4 signaling and inflammatory responses.

Leishmania donovani amastigote component-induced colony-stimulating factor production by macrophages: modulation by morphine

The neuroimmunomodulatory effects of opiates during microbial infections are now well known; however, not much is known during leishmaniasis. Here, we report the effects of morphine on purified ~12-kDa component of Leishmania donovani amastigote antigen (LDAA-12)-induced colony-stimulating factor (CSF) production by mouse peritoneal macrophages (PMs) in vitro. Low concentrations (1 × 10 –9 and 1 × 10 –11 M) of morphine significantly ( P < 0.05) augmented the production of CSFs, whereas high concentrations (1 × 10 -3 and 1 × 10 –5 M) inhibited CSF production. Morphine exerted a similar concentration-dependent biphasic effect on the LDAA-12-induced elaboration of granulocyte (G)-macrophage (M)-CSF (GM-CSF) and M-CSF by PMs in their conditioned medium, as quantified by using enzyme-linked immunosorbent assay. Furthermore, selective agonists of μ-(DAGO) and δ-(DPDPE) opioid receptors also, respectively, augmented and inhibited the production of CSFs. Pretreatment of PMs with naloxone (1 × 10 –5 M) significantly ( P < 0.05) blocked the augmenting effect of morphine. In contrast, at 1 × 10 –5 M, naloxone lacked any effect on the inhibitory effect of morphine; however, its 100-fold higher concentration partially blocked it. This study, apparently for the first time, demonstrates that morphine, via surface opioid receptors, biphasically modulates the LDAA-12-induced CSF production by PMs, in vitro. These results thus show the implications of opiate abuse on the outcome of therapeutic interventions in areas where both visceral leishmaniasis and drug abuse are rampant.

Oxidized LDL modulates activation of NFkappaB in mononuclear phagocytes by altering the degradation if IkappaBs.

Oxidized low density lipoprotein (oxLDL) is known to alter the expression of inflammatory gene products in mononuclear phagocytes. The mechanisms involved in this effect were studied by examining the activation of nuclear factor kappaB (NFkappaB), a transcription factor known to be important in controlling the expression of such genes. Pretreatment of peritoneal macrophages with oxLDL modulated the activation of NFkappaB in response to either lipopolysaccharide (LPS) or the combination of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). In macrophages pretreated with oxLDL the nuclear translocation of Rel family members (RelA and c-Rel) is delayed (LPS) or markedly diminished (IFN-gamma/IL-2) and results in delayed or reduced appearance of kappaB binding activity within the nucleus. These changes in NFkappaB activation result from alterations in the stimulus-dependent degradation of IkappaB alpha and IkappaB beta. The effects of oxLDL on NFkappaB activation depend both on the degree of LDL oxidation (most potent with extensive oxidation) and on the time of exposure of the cells to the lipoprotein preparation (a minimal exposure of 6 h is required before inhibitory effects are observed). The modulation of IkappaB/NFkappaB function in cells exposed to oxLDL appears to be responsible for previously reported effects of oxLDL on chemoattractant cytokine gene expression where both inhibition and delay of such stimulus-dependent events has been observed.

Functional Properties of Murine Macrophages Promoted by Nerve Growth Factor

The stimulating effect of nerve growth factor (NGF) on phagocytosis, parasite killing, and interleukin-1beta (IL-1beta) production of murine peritoneal macrophages was assessed. In the presence of various doses of NGF, macrophages showed the increased phagocytosis of both nonspecific hydrophilic microspheres and sheep red blood cells (SRBC) opsonized with anti-SRBC antibodies (Ab) or complement in a dose-dependent manner. NGF also enhanced killing of Leishmania donovani promastigotes by macrophages, and its ability was comparable with that of an optimal dose of recombinant granulocyte-macrophage colony-stimulating factor or recombinant interferon-gamma. The addition of NGF to peritoneal macrophages and monocyte-macrophage J774A.1 cells led to a significant release of IL-1beta in a dose-dependent manner and expression of IL-1beta mRNA. Because pretreatment of peritoneal macrophages and J774A.1 cells with K-252a, a tyrosine kinase inhibitor, completely suppressed these NGF-mediated stimulating effects and p140trk phosphorylation and because flow cytometric analysis with specific Ab against two distinct NGF receptors showed the expression of p140trk, unlike p75LNGFR, on the surface of macrophages, the stimulating activity of NGF to murine macrophages may be mediated through p140trk. Thus, NGF may act as an activator for murine macrophages in the process of inflammatory and immune actions.

Effect of Nickel Sulfide on Induction of Interleukin-1 and Phagocytic Activity

Rat peritoneal macrophages were treated with crystalline nickel sulfide (NiS), washed, and then tested for interleukin-1 (IL-1) production and phagocytosis of IgG-opsonized sheep red blood cells (SRBC). Pretreatment of peritoneal macrophages with crystalline NiS did not affect their ability to produce IL-1. In contrast, pretreatment of peritoneal macrophages with crystalline NiS inhibited the phagocytic activity of the peritoneal macrophage. This inhibitory activity was due to an effect on the internalization step of the phagocytic process. The binding step of this process was not affected by exposure to crystalline NiS. The overall number of Ig-SRBC that interacted with each macrophage was not different in treated or untreated cultures. This interaction is mediated through Fc receptors on the macrophage membrane. This result suggests that crystalline NiS does not modulate the expression of Fc receptors by peritoneal macrophages. The results described in this study suggest that crystalline NiS has a potent immunomodulatory effect on macrophage phagocytic activity.

Serum components induce beta-D-glucan-inhibitable uptake of zymosan particles by murine peritoneal macrophages.

Effects of murine serum (NMS) treatment on (1-->3)-beta-D-glucan inhibitable uptake of zymosan particles (ZYM) (GIZUP) by murine peritoneal macrophages (PM) and the structural specificity of the inhibition were examined. ZYM uptake by PM treated with NMS was enhanced in comparison with those treated with medium, and in a concentration- and incubation time-dependent manner. The enhanced ZYM uptake was significantly reduced by the pretreatment of PM with soluble (1-->3)-beta-D-glucans. These facts suggest that NMS enhances GIZUP. The effect disappeared by the treatment of NMS with gelatin-Sepharose which removed fibronectin (FN) from the serum, suggesting a significant contribution of FN on GIZUP. In addition, the administration of beta-glucan in vivo elevated the concentration of FN in serum by acute phase response and enhanced GIZUP, suggesting the positive contribution of acute phase responses on beta-glucan mediated immunopharmacological activities. Of particular interest, the inhibition was shown by both antitumor active and inactive glucans. These facts suggested that the recognition of beta-glucans by PM, which would proceed at a relatively early period of whole activation pathways, would not be enough to fully activate the host to show antitumor activity.

Early interactions of herpes simplex virus with mouse peritoneal macrophages.

Adsorption of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to resident peritoneal macrophages (PM) of 4-week-old Swiss albino (SA) and GR/AFib mice was studied. A significantly (P less than 0.05) higher HSV-2 adsorption rate was found with PM of SA mice than with PM of GR/AFib mice. Of added HSV-2 65% bound to the cells of SA mice over a 120-min period versus 15% to PM of GR/AFib mice. Only 15 to 20% of added HSV-1 bound to PM regardless of the mouse strain. These patterns of adsorption were found with all four HSV-1 and four HSV-2 strains tested. Pretreatment of PM with an HSV-2 mutant blocked the adsorption of added HSV-2. Thus, the receptors for HSV attachment seemed to be virus type selective. To avoid masking of adsorption by phagocytotic activity, the adsorption studies had to be performed at 4 degrees C. Transport of attached HSV-1 and HSV-2 to the nuclei of SA PM was studied with purified virus labeled with 32Pi and [3H]thymidine. In double-isotope experiments, only transport of HSV-2 was detected. The possible importance of differences in density or avidity of virus-binding receptors on the plasma membrane of PM is discussed in relation to macrophage-dependent focal liver necrosis, which was only demonstrable after intraperitoneal inoculation of HSV-2, not HSV-1, only in SA, not GR/AFib, mice.